comparative genome analysis of the spl gene family reveals
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Comparative genome analysis of the SPL gene family reveals novelevolutionary features in maize
Xiaojian Peng1*, Qianqian Wang2*, Yang Zhao1, Xiaoyu Li1 and Qing Ma1
1National Engineering Laboratory of Crop Stress Resistance, Key Laboratory of Crop Biology of Anhui
Province, School of Life Sciences, Anhui Agricultural University, Hefei, China.2 Institute of Horticulture, Anhui Academy of Agricultural Sciences, Hefei, China.
Abstract
SPLs are plant-specific transcription factors that play important regulatory roles in plant growth and development.Systematic analysis of the SPL family has been performed in numerous plants, such as Arabidopsis, rice, andPopulus. However, no comparative analysis has been performed across different species to examine evolutionaryfeatures. In this study, we present a comparative analysis of SPLs in different species. The results showed that 84SPLs of the four species can be divided into six groups according to phylogeny. We found that most of theSPL-containing regions in maize showed extensive conservation with duplicated regions of rice and sorghum. Agene duplication analysis in maize indicated that ZmSPLs showed a significant excess of segmental duplication. TheKa/Ks analysis indicated that 9 out of 18 duplicated pairs in maize experienced positive selection, while SPL genepairs of rice and sorghum mainly evolved under purifying selection, suggesting novel evolutionary features forZmSPLs. The 31 ZmSPLs were further analyzed by describing their gene structure, phylogenetic relationships, chro-mosomal location, and expression, Among the ZmSPLs, 13 were predicated to be targeted by miR156s and involvedin drought stress response. These results provide the foundation for future functional analyses of ZmSPLs.
Keywords: SPL, phylogenetic relationship, gene duplication, miR156 expression.
Received: June 8, 2017; Accepted: October 4, 2018.
Introduction
Transcription factors (TFs) are a large class of regula-
tors controling gene expression by activating or repressing
target genes at the transcriptional level. Increasing evi-
dence indicates that TFs have important roles in the regulat-
ing networks of plant growth and development processes
(Riechmann et al., 2001). SPLs (SQUAMOSA promoter
binding protein-like) comprise a family of plant-specific
transcription factors that contain a highly conserved SBP
domain consisting of about 76 amino acids (Chen et al.,
2010). This domain has been implicated in DNA binding
and nuclear localization, and also features two zinc-binding
sites assembled as Cys-Cys-Cys-His and Cys-Cys-His-
Cys, respectively (Klein et al., 1996; Yamasaki et al.,
2004). Gene structure analysis indicated that the nuclear lo-
calization signal (NLS) region partially overlapped with
the second Zn-finger located at the C-terminal of the SBP
domain. SBP-domain encoding proteins were firstly iso-
lated from Antirrhinum majus designated as AmSBP1 and
AmSBP2. These two proteins can recognize a conserved
motif in the promoter region of the floral meristem identity
gene SQUAMOSA, which is a member of the MADS-box
gene family based on its in vitro binding activity (Klein et
al., 1996). Subsequent experiments indicated that the palin-
dromic GTAC core motif of the cis-element is essential for
efficient DNA binding by different SBP proteins (Bir-
kenbihl et al., 2005; Cardon et al., 1997). To date, the SPL
gene family has been identified in various plant genomes,
such as Arabidopsis, rice, and Populus (Cardon et al., 1999;
Xie et al., 2006; Guo et al., 2008; Li and Lu 2014).
In Arabidopsis, a total of 16 members have been iden-
tified as SPL proteins. Several biological experiments dem-
onstrated that SPL proteins have important functions in
plant development processes, especially flower develop-
ment. For example, the AtSPL3 gene was shown to be in-
volved in the floral transition, and it was the first SPL gene
identified in Arabidopsis. As an ortholog of SQUAMOSA,
AtSPL3 can interact with the promoter region of the floral
meristem identity gene APETALA1 (AP1), and constitutive
expression of this gene in Arabidopsis can result in an early
flowering phenotype (Cardon et al., 1997). Loss-of-
function mutation of the Arabidopsis SPL8 gene indicated
Genetics and Molecular Biology, 42, 2, 380-394 (2019)
Copyright © 2019, Sociedade Brasileira de Genética.
DOI: http://dx.doi.org/10.1590/1678-4685-GMB-2017-0144
Send correspondence to Qing Ma. National Engineering Labora-tory of Crop Stress Resistance, Key Laboratory of Crop Biology ofAnhui Province, School of Life Sciences, Anhui Agricultural Univer-sity, No. 130, Changjiang West Road, Hefei 230036, China. E-mail:maqingahau2016@163.com.*These authors contributed equally to this work.
Research Article
that AtSPL8 can regulate pollen sac development (Unte et
al., 2003). In maize, the tasselsheath4 (tsh4) mutant of an
SPL gene was shown to regulate bract development and the
establishment of meristem boundaries (Chuck et al., 2010).
In addition, SPL genes (SPLs) were also demonstrated to
play crucial roles in fruit development (Manning et al.,
2006), leaf development (Stone et al., 2005), plant hor-
mone signaling (Zhang et al., 2007), male fertility (Xing,
2010), and shoot development (Wu and Poethig, 2006).
Besides transcription factors, miRNAs are another
other class of important regulators of gene expression, act-
ing at the post-transcriptional level (Lee et al., 1993; Zhang
et al., 2006). These small RNA molecules (20-24 nucleo-
tides in length) can cause the degradation of mRNAs or re-
press translation by binding to the mRNAs of the target
genes (Zhang et al., 2006). Most of the miRNAs in plants
are evolutionarily conserved, encoded by gene families
(Jones-Rhoades et al., 2006). Among them, miR156/157, a
miRNA family that is highly conserved in plants (Axtell
and Bowman, 2008), is thought to be involved in important
developmental processes. Previous studies demonstrated
that half of the SPLs have been found to be targeted by
miR156/157 family. For example, 10 of the 16 Arabidopsis
SPLs, were targeted by the miR156 family (Rhoades et al.,
2002; Schwab et al., 2005; Wu and Poethig, 2006; Wang et
al., 2009; Yu et al., 2010). In rice, 11 of the 19 SPLs were
found to be regulated by OsmiR156 (Xie et al., 2006).
Despite the progress in function studies of SPLs in
many species, no comparative analysis has been reported
across different species to study the evolution and func-
tional relevance of this family. Although the maize SPL
gene family has been reported by Hultquist and Dorweiler
(Dorweiler, 2008), our understanding of this gene family in
maize is still rather limited. Therefore, we firstly performed
a comparative analysis of this family to dissect the evolu-
tionary features in different species, and 31 ZmSPLs were
further characterized, including gene structure, phylogen-
etic relationships, gene duplication, amongst others. Quan-
titative real-time PCR (RT-qPCR) analysis was performed
to examine the expression pattern of miR156 targeted
genes in different tissues and in response to drought stress.
These results contribute to a basic understanding of the SPL
gene family in different species, and provide a foundation
to further elucidate the SPL gene function in maize.
Material and Methods
Whole-genome identification and phylogeneticanalysis of SPLs
To identify maize SPL proteins, the Hidden Markov
Model (HMM) profile of the SBP domain (PF03110.7) re-
trieved from Pfam database (http://pfam.xfam.org/) (Finn
et al., 2006) was adopted as query against maize genome
database (http://www.maizesequence.org/index.html),
with an cutoff E-value of le-3. Sequences of Arabidopsis
and rice SPL proteins were also used to query against the
maize genome to identify all possible maize SPL proteins
(Cardon et al., 1999; Xie et al., 2006; Guo et al., 2008). The
candidate sequences that met the standards were confirmed
again by Pfam database and SMART (http://smart.embl-
heidelberg.de/) (Letunic, 2009). Finally, redundant se-
quences were removed manually after alignments using
MUSCLE software (Edgar, 2004). To identify sorghum
SPLs, the complete genome sequence of sorghum was ob-
tained (ftp://ftp.ensemblgenomes.org/pub/plants/release-
31/fasta/sorghum_bicolor/pep/), and the same method as
described above was adopted. To understand the evolution-
ary relationships of the SPL family, full-length sequences
of the SPL proteins were aligned using MUSCLE software.
A phylogenetic tree was constructed using MEGA v4.0
(Tamura, 2007) by the neighbor-joining (NJ) method with
1,000 bootstrap replicates.
Synteny analysis, gene duplication, and evolutionanalysis
Syntenic blocks among maize, rice, and sorghum
were evaluated by MCScan software (Wang et al., 2012)
and alignments with an E-value of 1e-5 were considered sig-
nificant matches. Then, the duplicated SPLs from these
syntenic blocks were identified using a Perl script, and the
relationships of the duplicated genes, including segmental
and tandem duplications, were finally visualized using Cir-
cos (http://circos.ca) (Krzywinski et al., 2009; Wang et al.,
2015). DnaSP v5.0 (Rozas et al., 2003) was used to esti-
mate the number of nonsynonymous substitutions per non-
synonymous site (Ka) and synonymous substitution per
synonymous site (Ks) of the duplicated genes. The Ka/Ks
ratios between duplicated genes were analyzed to deduce
the selection model. To obtain further insight into selection
pressure among duplicated gene pairs, a sliding window
analysis of the Ka/Ks ratios was conducted with the follow-
ing parameters: window size 150 bp and step size 9 bp. For
duplication time analysis, the Ks value was translated into
duplication time in million years based on a synonymous
mutation rate of � substitutions per synonymous site per
year, as T = Ks/2�10-6 million years ago (Mya) (�= 6.510-9
for grasses) (Gaut et al., 1996; Quraishi et al., 2011).
Sequence analysis and chromosomal locations ofZmSPL genes
Information regarding the exon number, open reading
frame (ORF) length, molecular weight (kDa), and iso-
electric point (pI) of maize SPL proteins were determined
by the Expasy program (http://www.expasy.org/tools/).
Gene structure was predicted through alignments of the
coding sequences (CDS) with corresponding genomic se-
quences using GSDS (http://gsds.cbi.pku.edu.cn/) (Hu et
al., 2015). Conserved motifs were investigated by MEME
(Multiple Expectation Maximization for Motif Elicitation)
(Bailey and Elkan, 1995) with the parameters used in our
Peng et al. 381
previous study (Zhao et al., 2011). The chromosome loca-
tion image was generated by MapInspect software
(http://www.plantbreeding.wur.nl/uk/soft-
ware_mapinspect.html) according to the starting positions
of ZmSPLs on the 10 chromosomes.
Prediction of ZmSPL genes targeted by miR156
To predict ZmSPLs regulated by miR156, the se-
quence of maize miR156 was first obtained from miRBase
(http://www.mirbase.org/) (Kozomara and Griffithsjones,
2010). Then, ZmSPLs targeted by miR156 were predicted
by searching the coding regions and 3’ UTRs of all SPLs
for complementary sequences to the maize miR156 se-
quence using psRNATarget server with default parameters
(http://plantgrn.noble.org/psRNATarget/?function=3)
(Dai and Zhao, 2011).
Expression pattern analysis using transcriptomedata
Transcriptome data of the genome-wide gene expres-
sion atlas of the maize inbred line B73 was used to elucidate
the expression pattern of ZmSPLs during different develop-
ment stages (Sekhon et al., 2013). A heat map was gener-
ated based on the FPKM (fragments per kilobase of exon
per million fragments mapped) values, which were initially
transformed by taking log2 (FPKM+1) and then loaded into
R and the Bioconductor program
(http://www.bioconductor.org/) (Ross and Robert, 2008).
Plant materials, stress treatments, RNA extraction,and RT-qPCR analysis
To examine the expression profile during different
developmental stages, four representative tissues, includ-
ing root, leaf, stem, and silk were collected from a life cycle
of the maize inbred line B73. For stress treatment, maize
seeds were surface-sterilized in 1 (v/v) Topsin-M (Rotam
Crop Sciences Ltd.) for 10 min, washed in deionized water,
and germinated on wet filter paper at 28 °C for 3 days. The
germinated seeds were transplanted to enriched soil (turf to
vermiculite in a ratio of 1:1) and grown in a greenhouse
with a 14-h light/10-h dark cycle at 28-30 °C. Drought
stress was performed by withholding watering at the three-
leaf stage of maize seedlings. The seedling leaves were col-
lected at 0, 1, 2, and 4 days after treatment with relative leaf
water content (RLWC) decreased to 98, 70, 60, and 58%,
which represented normal plants, slight, moderate, and se-
vere stresses, respectively. For all the stages, three biologi-
cal replicates were performed for each sample. For RNA
isolation, all the collected samples were extracted using
Trizol reagent (Invitrogen). To remove possible contami-
nating genomic DNA, the extracted RNAs were treated
with DNase I (Invitrogen) for 20 min, then cDNAs were
synthesized from 1 �g of total RNA using the PrimerScript
RT Master mix (TaKaRa). For RT-qPCR analysis, gene-
specific primers for maize SPL genes were designed using
Primer Express 3.0 software (Applied Biosystems), listed
in Table S1, and the PCR assays and data analysis were per-
formed as described previously (Peng et al., 2012).
Primer specificity were examined through the Primer
Blast at NCBI, and their efficiency was tested by ordinary
PCR. Amplification products were analyzed by agarose gel
electrophoresis, and each primer pair was seen to amplified
only one 100 bp product, which indicated that these primers
were suitable for RT-qPCR. The RT-qPCRs were per-
formed in an ABI 7300 Real-Time machine, with a total re-
action volume of 20 �L, containing SYBR Green Master
Mix reagent, cDNA sample, primers and RNase-free water.
The PCR run program was as follows: denaturation (95 °C
for 10 min), amplification and quantification (40 cycles of
95 °C for 15 s and 60 °C for 1 min), melting curve analysis
(60–95 °C, with a heating rate of 0.3 °C/s). The ZmActin
gene was used for data normalization, and for each sample
three technical replicates were performed. Relative expres-
sion levels were calculated using the comparative delta
delta cycle threshold (��ct) method. The SPSS 19.0 soft-
ware (http://www.spss.com.cn/) was used for statistical
analysis.
Results
SPL genes in different species
In previous studies, a total of 19, 16, and 31 SPLs
were identified in rice, Arabidopsis, and maize, repectively
(Cardon et al., 1999; Xie et al., 2006; Dorweiler, 2008; Guo
et al., 2008). Due to maize genome database updates, we
performed a BlastP search against the genome database to
identify maize SPLs using the Hidden Markov Model
(HMM) profile of the SPL domain, and the same strategy
was used to identify sorghum SPLs. By this approach, a to-
tal of 31 and 18 non-redundant sequences in maize and sor-
ghum were identified after searching against Pfam and
SMART, respectively. The total number of SPLs in maize
was the same as in a previous study. In addition, the number
of sorghum SPLs was similar to that in rice and
Arabidopsis. These genes were named ZmSPL1–ZmSPL31
and SbSPL1–SbSPL18 according to their order of distribu-
tion on the chromosomes (Tables S2, S3). It should be
noted that the number of SPLs in the maize genome was
greater than that in rice, Arabidopsis, and sorghum. This
gives rise to the question, as to where did these additional
genes originally come from in the maize genome. To eluci-
date the possible mechanism(s) of this phenomenon, we
subsequently performed a comparative analysis of SPL
gene family in these species.
Phylogenetic relationships of SPLs
To examine the evolutionary relationships of SPLs
among different plant species, full-length sequences of the
SPL proteins were aligned using MUSCLE, and then a
combined phylogenetic tree of 84 SPL protein sequences
382 Novel evolutionary features of maize SPL genes
from the four species, including 31 of maize, 19 of rice, 18
of sorghum, and 16 of Arabidopsis, was constructed using
the NJ method with 1000 bootstrap replicates (Figure 1).
The 84 SPLs were divided into six subfamilies (I-VI) ac-
cording to phylogenetic relationship (bootstrap value >
50%). Although each of the subfamilies contained repre-
Peng et al. 383
Figure 1 - Phylogenetic relationships of maize, rice, sorghum, and Arabidopsis SPL proteins. The phylogenetic tree was constructed using MEGA4.0
with the NJ method. Bootstrap values above 50% are shown at each node.
sentative of rice, sorghum, and Arabidopsis SPLs, most
maize SPLs showed closer relationships with sorghum
SPLs than rice and Arabidopsis, suggesting a closer evolu-
tionary relationship of the two species. For example, a total
of 16 orthologous pairs were identified between maize and
sorghum. We noted that the number of SPLs located in dif-
ferent subfamilies had a significant difference, ranging
from 3 (III) to 20 (IV). Most of the members located in the
same phylogenetic clade had well-supported bootstrap val-
ues, while some proteins showed unclear evolutionary rela-
tionships with lower bootstrap values, such as AtSPL4,
AtSPL5, and AtSPL6. We also noted that the numbers of
maize SPL proteins in most of the six groups were higher
than other species, suggesting SPLs had especially ex-
panded in the maize genome.
Synteny analysis of SPLs among maize, sorghum,and rice
To examine the origin and evolutionary history of
SPLs among maize, sorghum, and rice, a comparative anal-
ysis was performed to identify SPL orthologous pairs. Be-
cause Arabidopsis belongs to the Dicotyledoneae group of
plants, orthologous pairs were not detected with the three
other species. Through the comparative analysis of the
genomic regions hosting the SPLs using MCScan software,
we observed strongly conserved synteny among the three
species. A total of 104 orthologous gene pairs were found
among maize, rice, and sorghum, including 38 pairs be-
tween maize and rice, 36 pairs between maize and sor-
ghum, and 30 pairs between sorghum and rice (Figure 2,
Table S4). The numbers of orthologous gene pairs among
384 Novel evolutionary features of maize SPL genes
Figure 2 - Synteny analysis of 68 SPLs from maize, sorghum, and rice. Maize, sorghum and rice chromosomes were labeled zm, sb, and os by different
color boxes, respectively. The numbers along each chromosome box indicate sequence length of each chromosome in megabases. Black lines represent
the syntenic relationships of orthologous gene pairs.
the three plants were similar, suggesting the conserved evo-
lution of the SPL family. Some differences were also ob-
served among the three species, for example, the ZmSPL16
and ZmSPL17 had two orthologous genes in rice
(ZmSPL16/OsSPL4, OsSPL11; ZmSPL17/OsSPL3,
OsSPL12), while only one was identified in sorghum
(ZmSPL16/SbSPL8; ZmSPL17/SbSPL7), respectively,
which might be related to gene loss in the evolution of sor-
ghum. In addition, the syntenic information also provided
important clues to study the putative function of the collin-
ear gene. For example, ZmSPL4 encoding the lg1 gene
(Moreno et al., 1997) had one collinear gene in rice
(OsSPL8) as well as in sorghum (SbSPL12). Especially,
ZmSPL11 encoding the tga1 gene (Wang et al., 2005) had
two orthologous genes in rice (OsSPL16 and OsSPL18) and
sorghum (SbSPL3 and SbSPL13). These genes existing in
different species might have originated from a common an-
cestor, which might share a similar regulatory role in plant
growth and development.
Gene duplication of SPLs
The number of ZmSPLs (31) was almost twice that of
Arabidopsis (16), and also much higher than that in rice
(19) and sorghum (18) (Cardon et al., 1999; Xie et al.,
2006). Gene duplication, including tandem and segmental
duplications, are thought to have played important roles in
the amplification of gene families in animals and plants
(Moore and Purugganan, 2003). Thus, potential duplication
events were analyzed to reveal the mechanism(s) behind
the expansion of the maize SPL family. According to the
syntenic regions and phylogenetic analysis, 18 ZmSPL
gene pairs (24 genes) were located on the segmental dupli-
cation regions, accounting for 77.4% of the number of
ZmSPLs (Figure 3, Table 1). In sorghum, six gene pairs
(nine genes) were localized on the segmental duplication
regions, accounting for 50% of the sorghum SPLs. In rice,
11 members forming seven gene pairs were detected, which
accounted for 57.8% of the rice SPLs. In addition, no signif-
icant tandem duplication events were detected among the
three plants. These findings indicated that segmental dupli-
cation was the major factor that contributed to the expan-
sion of SPL gene family, especially for maize.
To further understand the duplication and divergence
of SPLs, the Ka, Ks, and Ka/Ks ratio were calculated for
each duplicated pair. The Ka and Ks results were used to
examine the course of divergence after duplication, and the
Ka/Ks ratio was applied to explore different selective con-
strains. Generally, a Ka/Ks ratio < 1 means purifying selec-
tion, a ratio = 1 indicates neutral selection, while a ratio > 1
stands for positive selection (Lynch and Conery, 2000).
The results showed that the Ka/Ks ratio of the 18 duplicated
ZmSPLs pairs ranged from 0.449 to 1.605. Among them,
nine duplicated pairs had a Ka/Ks ratio <1. Moreover, the
values of ZmSPL13/-5, ZmSPL15/-22 and ZmSPL22/-24
were less than 0.6, which suggests strong purifying selec-
tion during evolution. The other nine pairs showed a Ka/Ks
ratio >1, indicating that these gene pairs evolved under pos-
itive selection (Table 1). In rice and sorghum, the Ka/Ks ra-
tios of all gene pairs were < 1, except for OsSPL2/-18,
suggesting that these gene pairs mainly evolved under puri-
fying selection. To obtain further insight into the selection
pressure of different sites/regions, we performed a slid-
ing-window analysis of the Ka/Ks ratio for each duplicated
gene pair. As shown in Figure 4, numerous sites/regions
showed evidence of strong positive selection, especially for
ZmSPL gene pairs. In contrast, the other sites/regions were
conserved under purifying selection, such as OsSPL14/-17
and SbSPL2/-15.
According to the estimation for Ks, the dates for 31
segmental duplication pairs of maize, rice, and sorghum,
were calculated based on a rate of 6.5 10-9 substitutions per
site per year (Gaut et al., 1996; Quraishi et al., 2011). The
results indicated that the 18 maize duplication events were
estimated to have occurred approximately between 4.81 to
Peng et al. 385
Figure 3 - Synteny analysis of maize (a), rice 9 (b), and sorghum (c) SPLs. Maize, sorghum, and rice chromosomes were labeled zm, sb and os by differ-
ent color boxes, respectively. The number along each chromosome box indicate sequence length of each chromosome in megabases. Black lines represent
the syntenic relationships between SPLs.
50.08 Mya (Table 1), and the duplication events of rice and
sorghum SPLs were estimated to have occurred between
34.46 to 47.00 Mya.
Sequence analysis of maize SPLs
Molecular weight (MW) and isoelectric point (pI) of
the 31 ZmSPLs were determined using the Expasy server.
The results showed that the ZmSPL proteins had a large
variation in the length (bp) of the open reading frame (rang-
ing from 300 to 3,339 bp) (Table S2). The 31 ZmSPLs were
divided into six subfamilies based on the unrooted NJ tree
(Figure S1a). Gene structure analysis indicated that the
maize SPL family had highly diverse distributions of exon
regions (Figure S1b). However, most SPLs within the same
subfamilies of the phylogenetic tree had a similar gene
structure. A total of 20 conserved motifs were identified in
the maize SPL proteins (Table S5). Compared with the
phylogenetic analysis, we found that genes located in the
same subfamily had similar motif compositions (Figure
S2). According to the starting positions of the maize SPL
genes annotated by the maize B73 genome database, chro-
mosome location analysis indicated that all of the 31
ZmSPLs were mapped to 9 of the 10 chromosomes with a
clear non-random distribution (Figure S3) with approxi-
mately 45% of the SPLs on chromosome 4 (eight genes)
and 5 (six genes).
Identification of ZmSPLs targeted by miR156
A series of SPLs have been confirmed to be targeted
by miR156 in Arabidopsis, grape, and Populus. In general,
the complementary sites of miR156 tend to be completely
conserved and to locate in the coding regions or 3’ UTRs of
386 Novel evolutionary features of maize SPL genes
Table 1 - Ka/Ks analysis and estimated divergence time for the duplicated SPL paralogs
Duplicated pairs Ka Ks Ka/Ks Purifying selection Date (Mya) Duplicate type
ZmSPL1-ZmSPL13 0.135 0.164 0.822 Yes 12.61 Segmental
ZmSPL5-ZmSPL25 0.122 0.126 0.966 Yes 9.68 Segmental
ZmSPL1-ZmSPL5 0.394 0.395 0.997 Yes 30.39 Segmental
ZmSPL1-ZmSPL25 0.374 0.344 1.088 No 26.45 Segmental
ZmSPL13-ZmSPL5 0.346 0.651 0.532 Yes 50.08 Segmental
ZmSPL13-ZmSPL25 0.373 0.279 1.337 No 21.45 Segmental
ZmSPL2-ZmSPL14 0.100 0.063 1.605 No 4.81 Segmental
ZmSPL3-ZmSPL18 0.083 0.073 1.136 No 5.64 Segmental
ZmSPL4-ZmSPL31 0.145 0.093 1.559 No 7.14 Segmental
ZmSPL6-ZmSPL11 0.480 0.565 0.849 Yes 43.49 Segmental
ZmSPL8-ZmSPL27 0.114 0.122 0.934 Yes 9.35 Segmental
ZmSPL9-ZmSPL29 0.124 0.100 1.242 No 7.65 Segmental
ZmSPL15-ZmSPL22 0.173 0.385 0.449 Yes 29.63 Segmental
ZmSPL22-ZmSPL24 0.365 0.611 0.598 Yes 46.98 Segmental
ZmSPL16-ZmSPL21 0.101 0.085 1.178 No 6.56 Segmental
ZmSPL17-ZmSPL20 0.257 0.272 0.948 Yes 20.88 Segmental
ZmSPL17-ZmSPL19 0.553 0.526 1.051 No 40.46 Segmental
ZmSPL20-ZmSPL19 0.440 0.394 1.118 No 30.30 Segmental
SbSPL2-SbSPL15 0.228 0.467 0.488 Yes 35.923 Segmental
SbSPL3-SbSPL6 0.406 0.534 0.760 Yes 41.08 Segmental
SbSPL3-SbSPL13 0.206 0.2061 0.474 0.435 Yes 34.46 Segmental
SbSPL6-SbSPL13 0.426 0.597 0.714 Yes 45.92 Segmental
SbSPL18-SbSPL7 0.265 0.611 0.433 Yes 47.00 Segmental
SbSPL17-SbSPL9 0.306 0.419 0.730 Yes 32.23 Segmental
OsSPL2-OsSPL16 0.380 0.519 0.732 Yes 39.92 Segmental
OsSPL2-OsSPL18 0.496 0.496 0.496 1.000 No 38.15 Segmental
OsSPL3-OsSPL12 0.487 0.524 0.929 Yes 40.31 Segmental
OsSPL4-OsSPL11 0.317 0.535 0.593 Yes 41.15 Segmental
OsSPL5-OsSPL10 0.338 0.412 0.820 Yes 31.69 Segmental
OsSPL14-OsSPL17 0.185 0.450 0.411 Yes 34.62 Segmental
OsSPL16-OsSPL18 0.292 0.461 0.633 Yes 35.46 Segmental
Peng et al. 387
Figure 4 - Sliding window plots of segmental duplicated SPLs. Window size is 150 bp, and step size is 9 bp.
SPLs in different plants (Schwarz et al., 2008; Hou et al.,
2013; Li and Lu, 2014;). To identify the ZmSPLs targeted
by miR156, we searched the coding regions and 3’ UTRs of
all ZmSPLs for targets of maize miR156 using the
psRNATarget online prediction tool with default parame-
ters (Dai and Zhao, 2011). A total of 13 ZmSPLs were pre-
dicted to be potential targets of miR156 (Figure 5). We also
found that the targeting sites of miR156 were located in
coding regions for 11 ZmSPLs, and only two complemen-
tary sites were located in the 3’ UTRs (ZmSPL7 and
ZmSPL26). Consistent with previous studies, the targeting
sites of maize SPLs were highly conserved in the evolution
by the alignments of miR156 with their complementary se-
quence of maize SPLs (Figure 6).
Expression patterns of ZmSPL genes in differentdevelopmental stages
The transcriptome data of the genome-wide gene ex-
pression atlas of maize was used to analyze the expression
patterns of SPLs in different developmental stages (Sekhon
et al., 2013) (Figure 7). The results showed that most
ZmSPLs had ubiquitously expression in the 18 different tis-
sues. The group IV members seem to play regulatory roles
in maize at multiple development stages based on the con-
stitutive expression at relatively high level in all of the 18
tissues. On the contrary, the group I genes were only ex-
pressed in one or a few tissues and at a very low expression
level, for example, ZmSPL22 and ZmSPL31 are merely ex-
pressed in V3_Stem and SAM. Furthermore, ZmSPL15
was not expressed among the 18 tissues. By comparing the
expression patterns of the duplicated gene pairs, we found
that most of the duplicated gene pairs had similar expres-
sion patterns, but some with obvious divergence were also
observed. For example, ZmSPL31 is only expressed in
V3-Stem and SAM, while its paralog ZmSPL4 is expressed
in V3-Stem and SAM, different stages of leaf and 10-DAP
whole seed.
The expression patterns of the 13 ZmSPLs targeted by
miR156 were further investigated by quantitative real-time
PCR (RT-qPCR) in different tissues. Four representative
tissues, including root, leaf, stem, and silk were used in this
study. A total of 12 genes were detected in the four tissues
(ZmSPL12 was not detected), and different expression lev-
els were found. Most of the genes showed high expression
in stem or leaves, especially ZmSPL5, ZmSPL7, ZmSPL9,
ZmSPL10, and ZmSPL13. We also noted that segment
duplicated genes had similar expression patterns of, for ex-
ample ZmSPL5 and ZmSPL13, suggesting conserved evo-
lution in maize (Figure 8).
Expression patterns of ZmSPL genes under droughtstress
While most studies so far focused on divergent bio-
logical processes regulated by SPL genes, increasing evi-
dence indicates that SPLs have also important roles in the
response to abiotic stresses (Hou et al., 2013; Wang et al.,
2009). To identify the possible members of ZmSPLs in-
volved in drought stress, the expressions of the 13 miR156
targeted genes were further examined by RT-qPCR in
maize leaves under slight, moderate, and severe stress (Fig-
ure 9). Consistent with the results of the expression at dif-
ferent developmental stages, the expression of ZmSPL12
was not detected, and all of the other 12 genes were respon-
sive to drought stress, suggesting important functions in
stress regulation. Among the 12 genes, the highest expres-
sion level was observed under severe stress treatment, espe-
388 Novel evolutionary features of maize SPL genes
Figure 5 - ZmSPLs targeted by miR156. Open reading frames (ORFs) are
indicated by grey rectangles, the SBP domain is shown by blue rectangles,
and the lines flanking ORFs represent 3’ UTRs. miR156 targeting sites are
indicated by yellow rectangles.
Figure 6 - Sequence alignments of maize miR156 with their complemen-
tary sequence in the coding sequences and 3’ UTRs of ZmSPLs.
cially for ZmSPL10, -13, -21, and -26. In addition, the
segment with duplicated genes showed similar expression
patterns, which might suggest their redundant function in
the regulation of maize drought response.
Discussion
SPLs encode a large gene family of plant-specific
transcription factors that play crucial roles in plant growth
and development (Klein et al., 1996; Cardon et al., 1997).
In the present study, we performed a comparative analysis
of the SPL family to examine the evolutionary history in
different species, thus providing a foundation for gene
function analysis. At least 16 SPLs were reported in
Arabidopsis, 19 in rice, and 28 in Populus (Cardon et al.,
1999; Xie et al., 2006; Li and Lu, 2014). In this study, a to-
tal of 31 and 18 SPLs were identified in maize and sor-
ghum, respectively. The phylogenetic tree of the 84 SPL
proteins, including 31 of maize, 19 of rice, 18 of sorghum,
and 16 of Arabidopsis, were divided into six groups. It
should be noted that the number of maize SPLs was much
higher than that in the mentioned species. With the purpose
of elucidating the expansion mechanism of the maize SPL
family, gene duplication events were investigated, which
are thought to have occurred during the process of evolu-
tion. Generally, gene duplications were major driving for-
ces in the evolution of genomes, and played vital roles in
the expansion of gene families in various species (Moore
and Purugganan, 2003; Mehan, 2004; Cannon et al., 2004),
such as NBS, HD-Zip, PHD, and others (Zhao et al., 2011;
Cheng et al., 2012; Wang et al., 2015).
According to the phylogenetic relationships and syn-
teny analysis, a total of 18 segmental duplicate gene pairs of
maize SPLs were identified, which accounted for 77.4% of
maize SPL family genes. However, only 50% and 57.8% of
the sorghum and rice SPLs, respectively, were detected to
be involved in segmental duplication. Among the 68 SPLs
of the three species, no tandem duplication events were de-
tected. Thus, the segmental duplication was largely respon-
sible for the expansion of SPL gene family. By comparing
the frequency of segmental duplication in the three species,
the segmental duplication of maize SPLs was seen to be
more prevalent than in the sorghum and rice genomes,
which provided a possible reason or explanation for why
the numbers of SPLs are significantly different among
maize, rice, and sorghum. In general, tandem duplication
often occurred in rapidly evolving gene families, while seg-
mental duplication was commonly reported in more slowly
evolving gene families, e.g. the HD-Zip gene family (Can-
non et al., 2004; Guo et al., 2008; Zhao et al., 2011). We
concluded that the prevalence of segmental duplication
Peng et al. 389
Figure 7 - Expression profiles of ZmSPLs at different developmental stages. Blue and red indicate low and high levels of transcript abundance, respec-
tively. Tissues from different developmental stages are shown at the bottom of the heat map.
demonstrated the slow evolutionary rate of the SPL gene
family. In fact, a total of 38 orthologous gene pairs were
identified between maize and rice, which was similar with
the result between maize and sorghum (36), as well as be-
tween rice and sorghum (30). Therefore, these results sug-
gested that the SPL gene family is a highly conserved and
slowly evolving family in plants.
Whole-genome duplication (WGD) played crucial
roles in plant diversification and evolution, and was often
accompanied by polyploidization and gene loss (Otto and
Whitton, 2000; Soltis et al., 2009). Previous studies
showed that grass species have undergone several rounds
of WGD. For example, maize experienced an ancient dupli-
cation prior to the divergence of grasses at approximately
50-70 Mya and a additional WGD at approximately 5 Mya,
which separated maize from sorghum (Gaut, 2002; Salse et
al., 2008; Schnable et al., 2009). The duplication time for
the 18 ZmSPL segmental duplication pairs ranged from
4.81 to 50.08 Mya. Among them, seven pairs showed a du-
plication time of less than 10 Mya. However, all the seg-
mental duplication events in the rice and sorghum genomes
were shown to have occurred between 34.46 to 47.00 Mya.
These results suggested that some segmental gene pairs of
maize SPLs are due to a recent duplication. In addition, se-
lection pressure analysis indicated that 50% of the maize
duplicated pairs evolved under positive selection. Unlike in
maize, SPL gene pairs of rice and sorghum mainly evolved
under purifying selection, indicating novel evolutionary
features of maize SPLs.
miR156 is one of the miRNA families that is highly
conserved and functions in diverse processes associated
with growth and development. It has been shown to medi-
ate posttranscriptional regulation for a subset of SPLs
through direct cleavage (Wu et al., 2009; Yu et al., 2010).
For example, previous studies have identified 10, 11, and
18 potential SPLs as the targets of miR156 in rice, Populus,
and tomato, respectively (Wu and Poethig, 2006; Xie et al.,
2006; Addoquaye et al., 2008; Schwarz et al., 2008; Li and
Lu, 2014). In this study, 13 of 31 ZmSPLs contained
miR156 recognition sites. It is noteworthy that ZmSPL1
and ZmSPL17 are not regulated by miR156, while their du-
plicated genes ZmSPL13 and ZmSPL20 are targets of
miR156. This finding suggested that some distinct regula-
tory mechanisms might exist in these duplicated genes. In
most cases, the miR156-regulated SPLs are master regula-
tors that play divergent and redundant roles in plant mor-
phology and development (Schwab, 2012). For example,
AtSPL3, AtSPL4, and AtSPL5 are mainly involved in the
390 Novel evolutionary features of maize SPL genes
Figure 8 - Expression patterns of 12 miR156 targeted ZmSPLs in four representative tissues. R: root; S: stem; L: leaf; F: filament. Shown are means � SE.
regulation of floral development (Cardon et al., 1997; Jung
et al., 2011), while AtSPL2, AtSPL10, and AtSPL11 have
been shown to be involved in lateral organ development in
the reproductive phase (Shikata et al., 2009). However,
whether the miR156-regulated ZmSPLs have similar regu-
latory roles remains to be further confirmed experimen-
tally.
According to the microarray expression profile analy-
sis, we found that some duplicated gene pairs have similar
expression patterns, suggesting that the duplicated genes
might have redundant functions in plant growth and devel-
opment. Exceptions to this were also observed. The phylo-
genetic analysis showed that most of the maize SPL
duplicated gene pairs located in the same branch had a high
bootstrap value, and the duplicated gene pairs also exhib-
ited similar exon/intron distribution and motif components.
However, some duplicated gene pairs were shown to have
significant divergence in expression patterns, such as
ZmSPL31 and ZmSPL4. These results suggested that most
of the duplicated gene pairs were still conserved in their
evolution, but that functional diversification has also ac-
companied the evolutionary process, as a major feature of
retained duplicated genes in long-term evolution (Blanc
and Wolfe, 2004). The expression patterns of the 12
miR156-targeted genes were further investigated at differ-
ent developmental stages by RT-qPCR. Among the 12
ZmSPLs, high expression was detected in leaf and stem. Es-
pecially, the results confirmed that some segment dupli-
cated genes have similar expression patterns, suggesting
their conserved evolution and redundant functions. The ex-
pression of the 12 ZmSPLs under drought stress was also
examined. Since most of the studies about SPL family were
related to developmental and biological processes, this re-
sult provided important information that the 12 miR156 tar-
geted genes are involved in drought stress, which may have
important implications in revealing the function and mech-
anism of SPL in the stress response.
With the advances of sequencing technologies, many
new miRNAs have been identified, and an increasing num-
ber of studies on miRNAs are being reported. miR156-
based regulation of SPL genes participates in various bio-
logical pathways and has been reported in many plants,
Peng et al. 391
Figure 9 - Expression patterns of 12 miR156 targeted ZmSPLs under drought stress. CK: normal plant; D1: slight stress; D2: moderate stress; D3: severe
stress. Shown are means � SE.
such as Arabidopsis, rice and others, but nearly no research
is reported in maize. Based on our experimental results, we
have identified several drought-response genes and cloned
them, and this will be further studied by transgenic technol-
ogy. In addition, we are verifying the actual regulatory rela-
tionship between miRNA156 and these cloned genes by 5’
RACE technology and degradation group sequencing tech-
nology, and we hope our research will reveal a new molecu-
lar mechanism in the maize abiotic stress response.
Acknowledgments
This research was supported by the Natural Science
Foundation of Anhui Province (NO. 1908085QC133 and
1908085QC134), the Science and Technology Major Pro-
ject of Anhui Province (NO. 18030701180), and the Na-
tional Natural Science Foundation of China (NO.
31540042 and 91435110). We thank the members of
bioinformatics group of the Key Laboratory of Crop Biol-
ogy of AnHui province for their assistance in this study.
Conflict of interest
The authors declare that they have no conflict of in-
terest.
Author contributions
XJP, QQW and QM conceived and designed the
study; XJP, QQW, YZ, XYL and QM conducted the exper-
iments; XJP, QQW, YZ and XYL analyzed the data; XJP,
QQW and QM wrote the manuscript; all authors read and
approved the final version.
References
Addoquaye C, Eshoo TW, Bartel DP and Axtell MJ (2008) En-
dogenous siRNA and miRNA targets identified by sequenc-
ing of the Arabidopsis degradome. Curr Biol 18:758.
Axtell MJ and Bowman JL (2008) Evolution of plant microRNAs
and their targets. Trends Plant Sci 13:343-349.
Bailey TL and Elkan C (1995) The value of prior knowledge in
discovering motifs with MEME. Proc Int Conf Intell Syst
Biol 3:21-29.
Birkenbihl RP, Jach G, Saedler H and Huijser P (2005) Functional
dissection of the plant-specific SBP-domain: Overlap of the
DNA-binding and nuclear localization domains. J Mol Biol
352:585-596.
Blanc G and Wolfe KH (2004) Widespread paleopolyploidy in
model plant species inferred from age distributions of dupli-
cate genes. Plant Cell 16:1667-1678.
Cannon SB, Mitra A, Baumgarten A, Young ND and May G
(2004) The roles of segmental and tandem gene duplication
in the evolution of large gene families in Arabidopsis tha-
liana. BMC Plant Biol 4:10.
Cardon GH, Höhmann S, Nettesheim K, Saedler H and Huijser P
(1997) Functional analysis of the Arabidopsis thaliana
SBP-box gene SPL3 : A novel gene involved in the floral
transition. Plant J 12:367–377.
Cardon G, Höhmann S, Klein J, Nettesheim K, Saedler H and
Huijser P (1999) Molecular characterisation of the
Arabidopsis SBP-box genes. Gene 237:91-104.
Cheng Y, Li X, Jiang H, Wei M, Miao W, Yamada T and Ming Z
(2012) Systematic analysis and comparison of nucleotide-
binding site disease resistance genes in maize. FEBS J
279:2431–2443.
Chuck G, Whipple C, Jackson D and Hake S (2010) The maize
SBP-box transcription factor encoded by tasselsheath4 reg-
ulates bract development and the establishment of meristem
boundaries. Development 137:1243-1250.
Dai X and Zhao PX (2011) psRNATarget: A plant small RNA tar-
get analysis server. Nucleic Acids Res 39:W155.
Dorweiler JE (2008) Feminized tassels of maize mop1 and ts1 mu-
tants exhibit altered levels of miR156 and specific SBP-box
genes. Planta 229:99-113.
Edgar RC (2004) MUSCLE: Multiple sequence alignment with
high accuracy and high throughput. Nucleic Acids Res
32:1792-1797.
Finn RD, Mistry J, Schusterböckler B, Griffiths-Jones S, Hollich
V, Lassmann T, Moxon S, Marshall M, Khanna A and
Durbin R (2006) Pfam: Clans, web tools and services. Nu-
cleic Acids Res 34: 247-251.
Gaut BS (2002) Evolutionary dynamics of grass genomes. New
Phytol 154:15–28.
Gaut BS, Morton BR, Mccaig BC and Clegg MT (1996) Substitu-
tion rate comparisons between grasses and palms: synony-
mous rate differences at the nuclear gene Adh parallel rate
differences at the plastid gene rbcL. Proc Natl Acad Sci USA
93:10274-110279.
Guo AY, Zhu QH, Gu X, Ge S, Yang J and Luo J (2008) Ge-
nome-wide identification and evolutionary analysis of the
plant specific SBP-box transcription factor family. Gene
418:1-8.
Hou H, Li J, Gao M, Singer SD, Wang H, Mao L, Fei Z and Wang
X (2013) Genomic organization, phylogenetic comparison
and differential expression of the SBP-box family genes in
grape. PLoS One 8:e59358.
Hu B, Jin J, Guo AY, Zhang H, Luo J and Gao G (2015) GSDS
2.0: An upgraded gene feature visualization server. Bioin-
formatics 31:1296.
Jones-Rhoades MW, Bartel DP and Bartel B (2006) MicroRNAs
and their regulatory roles in plants. Annu Rev Plant Biol
57:19-53.
Jung JH, Seo PJ, Kang SK and Park CM (2011) miR172 signals
are incorporated into the miR156 signaling pathway at the
SPL3/4/5 genes in Arabidopsis developmental transitions.
Plant Mol Biol 76:35-45.
Klein J, Saedler H and Huijser P (1996) A new family of DNA
binding proteins includes putative transcriptional regulators
of the Antirrhinum majus floral meristem identity gene
SQUAMOSA. Mol Genet Genomics 250:7-16.
Kozomara A and Griffiths-Jones S (2010) miRBase: Integrating
microRNA annotation and deep-sequencing data. Nucleic
Acids Res 39:D152-D157.
Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Hors-
man D, Jones SJ and Marra MA (2009) Circos: An informa-
tion aesthetic for comparative genomics. Genome Res
19:1639-1645.
392 Novel evolutionary features of maize SPL genes
Lee RC, Feinbaum RL and Ambros V (1993) The C. elegans
heterochronic gene lin-4 encodes small RNAs with anti-
sense complementarity to lin-14. Cell 75:843-854.
Letunic I (2009) SMART 6: Recent updates and new develop-
ments. Nucleic Acids Res 37:D229.
Li C and Lu S (2014) Molecular characterization of the SPL gene
family in Populus trichocarpa. BMC Plant Biol 14:131.
Lynch M and Conery JS (2000) The evolutionary fate and conse-
quences of duplicate genes. Science 290:1151-1155.
Manning K, Tör M, Poole M, Hong Y, Thompson AJ, King GJ,
Giovannoni JJ and Seymour GB (2006) A naturally occur-
ring epigenetic mutation in a gene encoding an SBP-box
transcription factor inhibits tomato fruit ripening. Nature
Genetics 38:948-952.
Mehan MR (2004) A genome-wide survey of segmental duplica-
tions that mediate common human genetic variation of chro-
mosomal architecture. Hum Genomics 1:335-344.
Moore RC and Purugganan MD (2003) The early stages of dupli-
cate gene evolution. Proc Natl Acad Sci USA 100:15682-
15687.
Moreno MA, Harper LC, Krueger RW, Dellaporta SL and Free-
ling M (1997) liguleless1 encodes a nuclear-localized pro-
tein required for induction of ligules and auricles during
maize leaf organogenesis. Genes Dev 11:616-628.
Otto SP and Whitton J (2000) Polyploid incidence and evolution.
Annu Rev Genet 34:401-437.
Peng X, Zhao Y, Cao J, Zhang W, Jiang H, Li X, Ma Q, Zhu S and
Cheng B (2012) CCCH-type zinc finger family in maize:
Genome-wide identification, classification and expression
profiling under abscisic acid and drought treatments. PLoS
One 7:e40120.
Quraishi UM, Abrouk M, Murat F, Pont C, Foucrier S, Desmai-
zieres G, Confolent C, Rivière N, Charmet G and Paux E
(2011) Cross-genome map based dissection of a nitrogen
use efficiency ortho-metaQTL in bread wheat unravels con-
certed cereal genome evolution. Plant J 65:745-756.
Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B and
Bartel DP (2002) Prediction of plant microRNA targets. Cell
110:513-520.
Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J,
Adam L, Pineda O, Ratcliffe OJ and Samaha RR (2001)
Arabidopsis transcription factors: Genome-wide compara-
tive analysis among eukaryotes. Science 290:2105-2110.
Ross I and Robert G (2008) R: A language and environment for
statistical computing. R Foundation for Statistical Com-
puting. Computing 1:12-21.
Rozas J, Sánchezdelbarrio JC, Messeguer X and Rozas R (2003)
DnaSP, DNA polymorphism analyses by the coalescent and
other methods. Bioinformatics 19:2496-2497.
Salse JM, Bolot S, Throude ML, Jouffe V, Piegu BT, Quraishi
UM, Calcagno T, Cooke R, Delseny M and Feuilleta C
(2008) Identification and characterization of shared duplica-
tions between rice and wheat provide new insight into grass
genome evolution. Plant Cell 20:11-24.
Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S,
Liang C, Zhang J, Fulton L and Graves TA (2009) The B73
maize genome: Complexity, diversity, and dynamics. Sci-
ence 326:1112-1115.
Schwab R (2012) The roles of miR156 and miR172 in phase
change regulation. In: Sunkar R (ed) MicroRNAs in Plant
Development and Stress Responses. Springer, Berlin, pp
49-75.
Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M and
Weigel D (2005) Specific effects of microRNAs on the plant
transcriptome. Dev Cell 8:517-527.
Schwarz S, Grande AV, Bujdoso N, Saedler H and Huijser P
(2008) The microRNA regulated SBP-box genes SPL9 and
SPL15 control shoot maturation in Arabidopsis. Plant Mol
Biol 67:183-195.
Sekhon RS, Briskine R, Hirsch CN, Myers CL, Springer NM,
Buell CR, Leon ND and Kaeppler SM (2013) Maize gene at-
las developed by RNA sequencing and comparative evalua-
tion of transcriptomes based on RNA sequencing and micro-
arrays. PLoS One 8:e61005.
Shikata M, Koyama T, Mitsuda N and Ohme-Takagi M (2009)
Arabidopsis SBP-box genes SPL10, SPL11 and SPL2 con-
trol morphological change in association with shoot matura-
tion in the reproductive phase. Plant Cell Physiol
50:2133-2145.
Soltis DE, Albert VA, Leebens-Mack J, Bell CD, Paterson AH,
Zheng C, Sankoff D, Depamphilis CW, Wall PK and Soltis
PS (2009) Polyploidy and angiosperm diversification. Am J
Bot 96:336-348.
Stone JM, Liang X, Nekl ER and Stiers JJ (2005) Arabidopsis
AtSPL14 , a plant-specific SBP-domain transcription factor,
participates in plant development and sensitivity to fumo-
nisin B1. Plant J 41:744-754.
Tamura K (2007) MEGA4: Molecular Evolutionary Genetics
Analysis (MEGA) software version 4.0. Mol Biol Evol.
24:1596-1599.
Unte US, Sorensen AM, Pesaresi P, Gandikota M, Leister D,
Saedler H and Huijser P (2003) SPL8, an SBP-box gene that
affects pollen sac development in Arabidopsis. Plant Cell
15:1009-1019.
Wang H, Nussbaumwagler T, Li B, Zhao Q, Vigouroux Y, Faller
M, Bomblies K, Lukens L and Doebley JF (2005) The origin
of the naked grains of maize. Nature 436:714-719.
Wang JW, Czech B and Weigel D (2009) miR156-regulated SPL
transcription factors define an endogenous flowering path-
way in Arabidopsis thaliana. Cell 138:738-749.
Wang Q, Liu J, Wang Y, Zhao Y, Jiang H and Cheng B (2015)
Systematic analysis of the maize PHD-finger gene family
reveals a subfamily involved in abiotic stress eesponse. Int J
Mol Sci 566:95-108.
Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, Lee TH, Jin
H, Marler B and Guo H (2012) MCScanX: a toolkit for de-
tection and evolutionary analysis of gene synteny and colli-
nearity. Nucleic Acids Research 40:e49
Wu G, Park MY, Conway SR, Wang JW, Weigel D and Poethig
RS (2009) The sequential action of miR156 and miR172
regulates developmental timing in Arabidopsis. Cell
138:750-759.
Wu G and Poethig RS (2006) Temporal regulation of shoot devel-
opment in Arabidopsis thaliana by miR156 and its target
SPL3. Development 133:3539-3547.
Xie K, Wu C and Xiong L (2006) Genomic organization, differen-
tial expression, and interaction of SQUAMOSA promoter-
binding-like transcription factors and microRNA156 in rice.
Plant Physiol 142:280-293.
Peng et al. 393
Xing SP (2010) miR156-targeted and nontargeted SBP-Box tran-
scription factors act in concert to secure male fertility in
Arabidopsis. Plant Cell 22: 3935-3950.
Yamasaki K, Kigawa T, Inoue M, Tateno M, Yamasaki T, Yabuki
T, Aoki M, Seki E, Matsuda T and Nunokawa E (2004) A
novel zinc-binding motif revealed by solution structures of
DNA-binding domains of Arabidopsis SBP-family tran-
scription factors. J Mol Biol 337:49-63.
Yu N, Cai WJ, Wang S, Shan CM, Wang LJ and Chen XY (2010)
Temporal control of trichome distribution by
microRNA156-targeted SPL genes in Arabidopsis thaliana.
Plant Cell 22:2322-2335.
Zhang B, Pan X, Cobb GP and Anderson TA (2006) Plant
microRNA: A small regulatory molecule with big impact.
Dev Biol 289:3-16.
Zhang Y, Schwarz S, Saedler H and Huijser P (2007) SPL8, a lo-
cal regulator in a subset of gibberellin-mediated develop-
mental processes in Arabidopsis. Plant Mol Biol 63:429-
439.
Zhao Y, Zhou Y, Jiang H, Li X, Gan D, Peng X, Zhu S and Cheng
B (2011) Systematic analysis of sequences and expression
patterns of drought-responsive members of the HD-Zip gene
family in maize. PLoS One 6:e28488.
Supplementary material
The following online material is available for this article:
Figure S1 - Phylogenetic relationships and gene structure
of the ZmSPLs.
Figure S2 - Distribution of conserved motifs identified in
the putative SPL proteins.
Figure S3 - Chromosomal locations of ZmSPLs on the 10
maize chromosomes.
Table S1 - List of gene-specific primers used in the present
study.
Table S2 - Detailed information on the 31 SPLs in the maize
genome.
Table S3 - Detailed information on the 18 sorghum SPLs.
Table S4 - Information about orthologous genes in maize,
rice, and sorghum.
Table S5 - Detailed information on the 20 motifs identified
in ZmSPLs.
Associate Editor: Adriana S. Hermely
License information: This is an open-access article distributed under the terms of theCreative Commons Attribution License (type CC-BY), which permits unrestricted use,distribution and reproduction in any medium, provided the original article is properly cited.
394 Novel evolutionary features of maize SPL genes
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